The present protocol describes a simple method for isolating preadipocytes from adipose tissue in broiler embryos. This method enables isolation with high yield, primary culture, and adipogenic differentiation of preadipocytes. Oil Red O staining and lipid/DNA stain measured the adipogenic ability of isolated cells induced with differentiation media.
Primary preadipocytes are a valuable experimental system for understanding the molecular pathways that control adipocyte metabolism. Chicken embryos provide the opportunity to isolate preadipocytes from the earliest stage of adipocyte development. This method has been optimized to yield cells with high viability and increased capacity to differentiate into mature adipocyte, which can be used for functional analysis of early life fat growth and development embryo in chicks.
The process of adipogenic differentiation is highly comparable between chickens and humans. Therefore, isolated preadipocytes can be used as a dual-proposed model for studies relevant to humans and poultry. To begin swab the embryo body with 70%ethanol.
Then to prevent interference during filtration at later steps after digestion gently scrubbed the skin surface with sterile gauze to remove feathers. Then cut off the skin between the legs and abdominal region to reveal the pair of femoral adipose depots. Use curved forceps with one hand to hold the skin around the leg and gently remove the femoral subcutaneous fat.
with the other hand. Later, use curved forceps to gently pull the depot away from the leg. Transfer the tissues into a 15 milliliter tube containing five milliliters of collection media and repeat the dissection for the other side of the fat pad.
Now, transfer the collected fat pads to a 60 millimeter Petri dish containing sterilization solution and rinse briefly by swirling the dish a few times. Then rinse the sterilization solution by transferring the fat pads to a 60 millimeter Petri dish containing PBS. Gently swirl the plate and once again, wash with PBS.
For digestion of the adipose tissues, transfer them to a 15 millimeter tube containing pre-warm enzymatic solution. Then immerse a pair of long straight scissors in the tube and finally mince the adipose tissues in the solution into pieces as small as possible. Transfer the minced tissue and enzymatic solution to a 25 milliliter autoclaved flask.
Wrap the flask with paraffin film and place the flask in an orbital shaker incubator at 37 degrees Celsius for digestion. After digestion, gently pipette up and down to mix well. Then remove any bits of undigested tissue and debris by filtering through a 250 micrometer tissue strainer into a 15 milliliter tube by pipetting.
Now, remove the cells adhered to the flask by rinsing the flask with four milliliters of growth media and filter the cell suspension into the same 15 milliliter tube. Next rinse the strainer by pipetting the growth media again to remove any cells trapped in the filter. Centrifuge at 300 times G for five minutes at room temperature to pellet the cell fraction and aspirate the supernatant without disturbing the cell pellet.
Gently resuspend the pellet in one milliliter of red blood cell lysis buffer, mix well by pipetting and incubate for five minutes at room temperature. Then dilute the cells by adding five milliliters of growth media to the tube containing the cells and mix them gently by pipetting. Now, pipette the cells onto a 40 micrometer cell strainer and filter them into a new 50 milliliter tube.
Then rinse the strainer with an additional five milliliters of growth media and pellet the cells by centrifuging at 300 times G for seven minutes at room temperature. Carefully aspirate the supernatant. Resuspend the remaining cell pellet in one milliliter of growth media by pipetting To determine the cell density and viability, mix 10 microliters of each sample in trypan blue by pipetting.
Load 10 microliters of the mixture onto the hemocytometer and count the cells. Analysis of preadipocytes after 24, 48, and 72 hours showed normal cell morphology and number. Adipogenic induction of preadipocytes showed rapid differentiation and accumulation of lipid droplets.
Aggressive handling of preadipocytes during isolation resulted in cell damage and low cell number. Microbial contamination can be observed despite taking preventative measures. Lipid staining with oil red O indicated the preadipocytes quickly begin to develop lipid droplets under adipogenic inducement and accumulation progresses over time as observed in 24, 48, and 72 hours.
The lipid accumulation relative to cell number was quantified using lipid and DNA stains. Both the lipid accumulation and cell proliferation increased over time. Low cell number or damaged cells can be observed when the tissue fractions are digested too long.
Therefore, it is recommended that one and a half hour of digestion is not exceeded. Isolated preadipocytes can be used for gene expression studies. Sufficient RNA can be obtained for use in cDNA synthesis and follow-on qPCR or RNAseq.
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